Bacterial persisters underlie many types of chronic bacterial infections and are a major public health problem. Bacterial persisters are a manifestation of bacterial individuality in which a subgroup of cells tolerates antibiotics, despit being genetically susceptible. The proposed research will develop techniques to isolate persisters in order to characterize their physiological state. To this end, a novel method for comprehensively analyzing population heterogeneity in protein abundances will be developed. This method will use large libraries of native, genomically-encoded proteins with C-terminal fluorescent tags for the important persistent pathogens E. coli, S. aureus, and P. aeruginosa. This method will also use developed culturing and fixing techniques with highly-automated flow cytometry to robustly analyze the protein abundance distributions for thousands of different proteins under conditions that increase persistence. These distributions will be analyzed to facilitate the rational pooling of fluorescently-tagged strains for a novel selection strategy to identify predictive proteins for persistence. The selection strategy uses fluorescently activated cell sorting (FACS) of the top fraction of cells with the highest protein abundances in a pooled library. The sorted cells will be treated with high concentrations of antibiotics in a persister assay, thereby enriching the frequency of cells harboring tagged proteins that predict persistence. After multiple iterations through the selection scheme, DNA- sequencing will be performed to determine the identity of the predictive tagged proteins. These protein markers will be validated by FACS and persister assays, and will be used to isolate pure persister populations. Powerful systems biology techniques, including RNA-seq and in vivo protein occupancy display, will be applied to pure cultures of persisters to characterize their physiology at the systems level. The developed techniques will provide deep insight into bacterial persistence and heterogeneity, and the findings will accelerate development of new antibiotic treatments.